中文摘要：

【目的】探讨反义RNA技术介导的大肠杆菌非必需基因rpsF基因沉默导致菌体生长受抑制的原因。【方法】将rpsF基因5’端41—230bp的片段反向插入带有末端配对结构的反义表达载体pHN678，获得重组质粒，导入大肠杆菌宿主获得反义RNA菌株Escherichia．coli／pHNF，并用诱导剂IPTG诱导反义RNA表达，通过与对照菌E．coli／pHN678的液体生长状态差异判断菌体生长表型；采用RealtimeRT—PCR方法跟踪分析转录水平。【结果】构建了针对rpsF的反义RNA菌株，且其生长受抑制程度与IPTG浓度呈正相关。IPTG浓度为100umol／L时，菌体生长未受抑制，但靶基因rpsF的mRNA量降低了36％，而rpsR是位于同一操纵子下游的必需基因，其转录水平却未受影响；IPTG浓度为200umol／L时，菌体生长明显受抑制，经分析发现rpsR转录水平降低了12％。【结论】反义RNA菌株E．coli／pHNF生长受抑制的原因是由于此反义RNA引起了同一操纵子下另一必需基因rpsR的转录水平降低。

英文摘要：

[ Objective] We explored the cause of cell growth inhibition by antisense RNA mediated nonessential gene silencing of rpsF gene in Escherichia coll. [ Methods] The 41 - 230 bp fragment around 5＇ end of gene rpsF was reversely cloned into antisense expression vector pHN678, which is flanked with a paired-termini. The recombinant plasmid was named pHNF. Then it was transformed into E. coli to produce antisense RNA strain E. coli/pHNF. Antisense RNA expression was induced by isopropyl-β-D-thiogalactopyranoside （IPTG） , the difference of liquid growth phenotype was identified between E. coli/pHNF and the control strain E. coli/pHN678; and gene transcriptional level was measured by Real time RT-PCR. [ Results l We obtained one antisense RNA strain targeted rpsF. We found that the reduced growth rate of this strain was positively related to the IPTG concentration. When IPTG was 100 txmol/L, the cell growth was not inhibited whereas the mRNA amount of rpsF had decreased by 36% , and mRNA of essential gene rpsR in the same operon did not decayed. However, when IPTG reached 200 txmol/L, the cell growth was obviously inhibited and rpsR mRNA was reduced by 12%. [ Conclusion ] The essential gene transcription level of rpsR decreases with the nonessential gene silencing of rpsF in the same operon, and leads to the growth inhibition of E. coli/pHNF.